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In recent years, microalgae have attracted considerable interest as a biofuel resource owing to their rapid growth, tolerance to harsh conditions, and ability to accumulate a large amount of triacylglycerols (TAGs). However, the economic effectiveness of algal biofuel is still low. In this study, we attempted to increase oil production of the microalga Scenedesmus quadricauda by elevating intracellular malonyl-CoA and glycerol-3-phosphate (G3P) pools. To increase intracellular oil content, yeast-derived genes encoding acetyl-CoA carboxylase (ACC1), glycerol kinase (GPD1), and glycerol-3-phosphate dehydrogenase (GUT1) were overexpressed under the control of CaMV 35S and NOS promoters with SV40 large T antigen components. Fatty acid profiling, G3P content, and the number of cells with high oil content were analyzed by gas chromatography-mass spectrometry, G3P assay kit, and flow cytometry, respectively. Overexpression of ACC1 increased the total fatty acid content by 1.6-fold. Overexpression of GPD1 and GUT1 increased intracellular G3P content by 1.6- and 1.9-fold, respectively. Multi-gene expression of ACC1, GPD1, and GUT1 increased the number of cells with high oil content by 1.45-fold compared with that observed with the wild-type. This study is the first to report increased oil production by overexpression of the key genes ( ACC1, GPD1, and GUT1) for TAG biosynthesis in microalgae. [ABSTRACT FROM AUTHOR]

Streptococcus thermophilus is a lactic acid bacterium and used as starter culture in the dairy industry, mainly in the manufacture of yoghurt, with Lactobacillus delbrueckii subsp . bulgaricus. It produces lactic acid as a major fermentation end product and some carbonyl compounds through sugar metabolism. The level of metabolites could be improved using molecular biotechnology. The genes of als, encoding α-acetolactate synthase (Als), the pflA, encoding pyruvate-formate lyase activating enzyme (PflA), and the adhB which encodes alcohol dehydrogenase (AdhB) of S. thermophilus NCFB2393 strain were amplified by polymerase chain reaction and separately cloned into the overexpression vector pNZ276 under the control of the lacA promoter. The strains were transformed individually with the constructed plasmids. Their abilities to generate important metabolites such as pyruvate, lactate, formate, acetaldehyde, acetoin, ethanol, and 2,3-butanediol in LM17 medium were analyzed using high-performance liquid chromatography. High level of 2,3-butanediol was obtained by overexpressing the als gene. The level of formate increased slightly by overexpressing the pflA gene. The overexpression of the adhB gene, on the other hand, resulted in a significant increase in the ethanol level. [ABSTRACT FROM AUTHOR]

Homeobox genes HOXA9 and MEIS1 are evolutionarily conserved transcription factors with essential roles in both hematopoiesis and leukemogenesis. They act as dominant cooperating oncoproteins that cause acute leukemias bearing MLL translocations and to a lesser extent T-cell acute lymphocytic leukemia (ALL) characterized by other gene fusions. Overexpression is associated with an adverse prognosis in adults. In childhood, the genes have only been investigated in leukemias bearing MLL translocations. The aim of this study was to determine whether overexpression extends to leukemic subtypes other than the MLL -positive subtype in childhood. We use quantitative real-time PCR methodology to investigate gene expression in 100 children with acute leukemias and compare them to those of healthy controls. We show that abnormally high HOXA9 and MEIS1 gene expression is associated with a variety of leukemic subtypes, including various maturation stages of B-cell ALL and cytogenetic types other than the MLL -positive population, thus suggesting that the genes are implicated in the development of a broad range of leukemic subtypes in childhood. In addition, we show that HOXA9 and MEIS1 overexpression are inversely correlated with relapse and overall survival, so the genes could become useful predictive markers of the clinical course of pediatric acute leukemias. [ABSTRACT FROM AUTHOR]

Zn(II)2Cys6 transcription factor genes encode transcription regulators that manage the infection potential and production of secondary metabolites such as toxins in fungi. In this study, a gene namedrolPthat encodes a putative Zn(II)2Cys6 transcription factor regulating leucinostatin production in the filamentous fungusPaecilomyces lilacinuswas characterized by a gene knockout approach. The deduced proprotein consists of 705 amino acids and is highly homologous to the Zn(II)2Cys6 transcription factor of the entomopathogenic fungusMetarhizium brunneum. Predictive analysis of the secondary structure of the proportion showed that it has two domains.Paecilomyces lilacinuscan produce nematotoxins leucinostatins A and B. Deletion ofrolPfrom theP. lilacinuswild-type strain Pl36-1 leads to the absence of leucinostatin A, while a large increase in the leucinostatin A level was achieved in therolPoverexpression strain Ov-Pl36-1. During the process of nematode infection,rolPshowed high expression levels at 48–72 h and peaked at 48 h. Bioassay tests confirmed the requirement ofrolPfor leucinostatin production. However, the root-knot second-stage juvenile-toxicity was minimized to 35.3% in ΔrolP; toxicity was 97.2% with Ov-Pl36-1 compared with 68.4% with the wild type strain Pl36-1. Interestingly, neither knockout nor overexpression of therolPgene affected the growth or sporulation ofP. lilacinus. Additionally, fungal nutrition and acidic media might stimulate therolPactivity of the wild type strain against nematodes. These findings suggested that therolPgene is required for the induction and production of leucinostatins and is considered a leucinostatin regulatory gene inP. lilacinus. [ABSTRACT FROM AUTHOR]

In this report we characterized the Arabidopsis ABI1 gene orthologue and Brassica napus gene paralogues encoding protein phosphatase 2C (PP2C, group A), which is known to be a negative regulator of the ABA signaling pathway. Six homologous B. napus sequences were identified and characterized as putative PP2C group A members. To gain insight into the conservation of ABI1 function in Brassicaceae, and understand better its regulatory effects in the drought stress response, we generated transgenic B. napus plants overexpressing A. thaliana ABI1. Transgenic plants subjected to drought showed a decrease in relative water content, photosynthetic pigments content and expression level of RAB18- and RD19A-drought-responsive marker genes relative to WT plants. We present the characterization of the drought response of B. napus with the participation of ABI1-like paralogues. The expression pattern of two evolutionarily distant paralogues, BnaA01.ABI1.a and BnaC07.ABI1.b in B. napus and their promoter activity in A. thaliana showed differences in the induction of the paralogues under dehydration stress. Comparative sequence analysis of both BnaABI1 promoters showed variation in positions of cis-acting elements that are especially important for ABA- and stress-inducible expression. Together, these data reveal that subfunctionalization following gene duplication may be important in the maintenance and functional divergence of the BnaABI1 paralogues. Our results provide a framework for a better understanding of (1) the role of ABI1 as a hub protein regulator of the drought response, and (2) the differential involvement of the duplicated BnaABI1 genes in the response of B. napus to dehydration-related stresses. [ABSTRACT FROM AUTHOR]

Gene overexpression is a widely used functional genomics approach in fungal biology. However, to date it has not been established in Zymoseptoria tritici which is an important pathogen of wheat ( Triticum species). Here we report a suite of Gateway® recombination compatible ternary expression vectors for Agrobacterium tumefaciens mediated transformation of Z. tritici . The suite of 32 vectors is based on a combination of four resistance markers for positive selection against glufosinate ammonium, geneticin, hygromycin and sulfonylurea; three constitutive Z. tritici promoters ( pZtATUB , pZtGAPDH and pZtTEF ) and a nitrogen responsive promoter ( pZtNIA1 ) for controlled expression of the open reading frames. Half of the vectors facilitate expression of proteins tagged with C-terminal EGFP. All 32 vectors allow high frequency targeting of the overexpression cassette into the Ku70 locus and complement the Ku70 gene when transformed into a Z. tritici ku70 null strain, thus circumventing additional phenotypes that can arise from random integration. This suite of ternary expression vectors will be a useful tool for functional analysis through gene overexpression in Z. tritici . [ABSTRACT FROM AUTHOR]

The phytopathogenic ascomycete Botrytis cinerea produces several secondary metabolites that have biotechnical significance and has been particularly used for S-(+)-abscisic acid production at the industrial scale. To manipulate the expression levels of specific secondary metabolite biosynthetic genes of B. cinerea with Agrobacterium tumefaciens-mediated transformation system, two expression vectors (pCBh1 and pCBg1 with different selection markers) and one RNA silencing vector, pCBSilent1, were developed with the In-Fusion assembly method. Both expression vectors were highly effective in constitutively expressing eGFP, and pCBSilent1 effectively silenced the eGFP gene in B. cinerea. Bcaba4, a gene suggested to participate in ABA biosynthesis in B. cinerea, was then targeted for gene overexpression and RNA silencing with these reverse genetic tools. The overexpression of bcaba4 dramatically induced ABA formation in the B. cinerea wild type strain Bc-6, and the gene silencing of bcaba4 significantly reduced ABA-production in an ABA-producing B. cinerea strain. [ABSTRACT FROM AUTHOR]

Increased expression of genes encoding multidrug resistance efflux pumps (MDR-EPs) contributes to antimicrobial agent and biocide resistance in Staphylococcus aureus . Previously identified associations between norA overexpression and spa type t002 meticillin-resistant S. aureus (MRSA), and a similar yet weaker association between mepA overexpression and type t008 meticillin-susceptible S. aureus (MSSA), in clinical isolates are suggestive of clonal dissemination. It is also possible that related strains are prone to mutations resulting in overexpression of specific MDR-EP genes. Exposure of non-MDR-EP-overexpressing clinical isolates to biocides and dyes can select for MDR-EP-overexpressing mutants. spa types t002 and t008 isolates are predominated by multilocus sequencing typing sequence types (STs) 5 and 8, respectively. In this study, non-MDR-EP gene-overexpressing clinical isolates (MRSA and MSSA) representing ST5 and ST8 were subjected to single exposures of ethidium bromide (EtBr) to select for EtBr-resistant mutants. Measurements of active EtBr transport among mutants were used to demonstrate an efflux-proficient phenotype. Using quantitative reverse-transcription PCR, it was found that EtBr-resistant mutants of ST5 and ST8 parental strains predominantly overexpressed mepA (100%) and mdeA (83%), respectively, regardless of meticillin sensitivity. Associations between clonal lineage and MDR-EP gene overexpression differed from those previously observed and suggest the latter is due to clonal spread of efflux-proficient strains. The predilection of in vitro-selected mutants of related strains to overexpress the same MDR-EP gene indicates the presence of a consistent mutational process. [ABSTRACT FROM AUTHOR]

The targeting of transgenic constructs at single copy into neutral genomic loci avoids the unpredictable outcomes associated with conventional random integration approaches. The Gt(ROSA)26Sor locus on chromosome 6 has been used many times for the integration of transgenic constructs and is known to be permissive for transgene expression and disruption of the gene is not associated with a known phenotype. Furthermore, the transcript made from the Gt(ROSA)26Sor locus is ubiquitously expressed and subsequently the locus can be used to drive the ubiquitous expression of transgenes.Here we report a protocol for the generation of targeted transgenic alleles at Gt(ROSA)26Sor, taking as an example a conditional overexpression allele, by PhiC31 integrase/recombinase-mediated cassette exchange of an engineered Gt(ROSA)26Sor locus in mouse embryonic stem cells. The overexpression allele is initially silenced by the presence of a loxP flanked stop sequence but can be strongly activated through the action of Cre recombinase., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background Ovarian carcinoma is one of the most common gynecological cancers with high mortality rates. Numerous evidences demonstrate that cancer cells undergo metabolic abnormality during tumorigenesis in tumor microenvironment and further facilitate tumor progression. Succinate dehydrogenase (SDH or Complex II) is one of the important enzymes in the tricarboxylic acid (TCA) cycle. Succinate dehydrogenase subunit B (SDHB) gene, which encodes one of the four subunits of SDH, has been recognized as a tumor suppressor. However the role of SDHB in ovarian cancer is still unclear. Methods Using the SDHB specific siRNA and overexpression plasmid, the expression of SDHB was silenced and conversely induced in ovarian cancer cell lines SKOV3 and A2780, respectively. The possible role of SDHB in ovarian cancer was investigated in vitro, using proliferation, migration and invasion assays. To explore the mechanism, proliferation and migration related proteins such as Bcl-2, cleaved caspase 3, p-ERK, MMP-2, and p-FAK were examined by western blot. P-P38, p-AMPKα, and HIF-1α were also examined by western blot. CoCl2 was used to induce HIF-1α expression in SKOV3 and A2780 cells. Results SDHB silencing promoted cell proliferation, invasion, and migration, but inhibited apoptosis of SKOV3 and A2780 cells. In contrast, overexpression of SDHB inhibited cell proliferation, invasion, migration, and promoted apoptosis in SKOV3 cells. It was observed that up-regulation of Bcl-2 and MMP-2, activation of p-P38, p-ERK, and p-FAK, inhibition of cleaved caspase 3 in SDHB-silenced cells. Meanwhile, decreased Bcl-2 and MMP-2, inhibition of p-P38, p-ERK, and p-FAK, activation of cleaved caspase 3 were shown in SDHB-overexpressed SKOV3 cells. HIF-1α, an essential factor in tumor progression, was up-regulated in SDHB-silenced cells with the activation of p-AMPKα and down-regulated in SDHB-overexpressed cancer cells with the decreased p-AMPKα. And SDHB was proved to be decreased due to upregulation of HIF-1α expression in CoCl2-treated cancer cells. Conclusions Our results firstly revealed that SDHB played a key role in cell proliferation, invasion, migration, and apoptosis of human ovarian carcinoma via AMPK-HIF-1α pathway. SDHB-overexpression might be a new approach to inhibit tumor progression in human ovarian carcinoma. [ABSTRACT FROM AUTHOR]

The advent of numerous technological platforms for genome sequencing has led to increasing understanding and construction of metabolic networks. A popular system engineering strategy is used to analyze microbial metabolic networks is flux balance analysis (FBA). In recent times, there has been a lot of interest in the study of the metabolic network dynamics when genes are overexpressed in the system. Herein, an optimization framework, which employs dynamic flux balance analysis (DFBA) is proposed for predicting ethanol concentration profiles in glycerol fermentations using Escherichia coli. In silico results were experimentally validated by overexpressing alcohol/acetaldehyde dehydrogenase adhE, pyruvate kinase pykF, pyruvate formate-lyase pflB and isoleucine-valine enzymes ilvC and llvL. [ABSTRACT FROM AUTHOR]

Reasoning that overexpression of multiple E2F-responsive genes might be a useful marker for RB1 dysfunction, we compiled a list of E2Fresponsive genes from the literature and evaluated their expression in publicly available gene expression microarray data of patients with breast cancer, serous ovarian cancer, and prostate cancer. In breast cancer, a group of tumors was identified, each of which simultaneously overexpressed multiple E2F-responsive genes. Seventy percent of these genes were concerned with cell cycle progression, DNA repair, or mitosis. These E2F-responsive gene overexpressing (ERGO) tumors frequently exhibited additional evidence of Rb/E2F axis dysfunction, were mostly triple negative, and preferentially overexpressed multiple basal cytokeratins, suggesting that they overlapped substantially with the basal-like tumor subset. ERGO tumors were also identified in serous ovarian cancer and prostate cancer. In these cancer types, there was no evidence for a tumor subset comparable to the breast cancer basal-like subset. A core group of about 30 E2F-responsive genes were overexpressed in all three cancer types. Thus, it appears that disorders of the Rb/E2F axis can arise at multiple organ sites and produce tumors that simultaneously overexpress multiple E2F-responsive genes. [ABSTRACT FROM AUTHOR]

Genomic duplications of varying lengths at Xq26-q27 involving SOX3 have been described in families with X-linked hypopituitarism. Using array-CGH we detected a 1.1 Mb microduplication at Xq27 in a large family with three males suffering from X-linked hypopituitarism. The duplication was mapped from 138.7 to 139.8 Mb, harboring only two annotated genes, SOX3 and ATP11C, and was shown to be a direct tandem copy number gain. Unexpectedly, the microduplication did not fully segregate with the disease in this family suggesting that SOX3 duplications have variable penetrance for X-linked hypopituitarism. In the same family, a female fetus presenting with a neural tube defect was also shown to carry the SOX3 copy number gain. Since we also demonstrated increased SOX3 mRNA levels in amnion cells derived from an unrelated t(X;22)(q27;q11) female fetus with spina bifida, we propose that increased levels of SOX3 could be a risk factor for neural tube defects. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Mycobacterium neoaurum NwIB-01 exhibits powerful ability to cleave the side chain of soybean phytosterols to accumulate 4-androstene-3,17-dione (AD) and 1,4-androstadiene-3,17-dione (ADD). The difficulty in separation of AD from ADD is one of the key bottlenecks to the microbial transformation of phytosterols in the industry. To enhance ADD quantity in products, 3-ketosteroid Δ-dehydrogenase genes ( kstD and kstD) were obtained from M. neoaurum NwIB-01 and Arthrobacter simplex respectively. Using replicating vector pMV261, kstD and kstD were overexpressed in M. neoaurum NwIB-01. For foreign gene stable expression, the integration vector pMV306 was used for kstD/ kstD overexpression and the relevant sequences of promoter and kanamycin antibiotic resistance gene sequences were amplified by PCR to verify plasmid integrity. The resultant plasmid and mutant strain were verified and the kstD augmentation mutants were good ADD-producing strains. The ADD producing capacity of NwIB-04 and NwIB-05 was 0.1401 and 0.1740 g/l (cultured in shake bottles with 0.4 g/l phytosterols), and the molar ratio of ADD in products was 98.34 and 98.60 %, respectively. This study on the manipulation of the main kstD gene in Mycobacterium sp. provides a feasible way to achieve excellent phytosterol-transformation strains with high product purity. [ABSTRACT FROM AUTHOR]

The role of the putative extracytoplasmic function sigma (σ) factor FujE, which has not been characterized as a member of the FK506 biosynthetic gene cluster, on FK506 production was identified by gene deletion, overexpression, and transcription analysis experiments in Streptomyces sp. strain KCCM 11116P. Inactivation of fujE had no effect on FK506 production, growth, or morphological differentiation. Overexpression of fujE with integrative vectors increased FK506 production by 2.87-fold (24.5 ± 1.4 mg·L-1) compared with the wild type (8.5 ± 0.5 mg·L-1). Semiquantitative reverse transcription - polymerase chain reaction analysis indicated that the overexpression of fujE stimulates the transcription of the FK506 biosynthetic genes. These results demonstrated that fujE is a new member of the FK506 biosynthetic gene cluster. [ABSTRACT FROM AUTHOR]

It is believed that hundreds of genes, including photosynthesis-related genes, are typically involved in plant response to aphid feeding. Up to now, however, it is little known on the relationship between the photosynthesis-related genes and plant response to herbivores. In this study, we identified a cotton photosynthesis-related gene ( GhPSAK1) which belongs to PSI-PSAK family and encodes a putative protein of 162 amino acids. RT-PCR analysis revealed that GhPSAK1 transcripts in leaves were increased at 12-24 h, but decreased at 48-72 h after cotton aphid attack or wounding induction. Choice assay and no-choice assay demonstrated that overexpression of GhPSAK1 in Arabidopsis improved plant tolerance to green peach aphids ( Myzus persicae). The defense response genes related to salicylic acid signaling pathway were enhanced in the GhPSAK1 overexpressing transgenic plants. In addition, the callose amount in transgenic Arabidopsis leaves was more than that of wild type. Contents of the soluble sugars and total amino acids were also altered in leaves of transgenic Arabidopsis plants. Activities of superoxide dismutase and peroxidase in transgenic leaves were higher than those of wild type. These results suggested that GhPSAK1 may be involved in regulation of cotton response and tolerance to aphid attack. [ABSTRACT FROM AUTHOR]

Highlights: [•] We tested a novel promoter of Gluconobacter oxydans, denominated gHp0169. [•] Use of gHp0169 promoter for heterologous and homologous gene expression was demonstrated. [•] The gHp0169 promoter exhibited a higher transcriptional activity than that of tufB promoter from G. oxydans. [ABSTRACT FROM AUTHOR]

Background Extensive studies using the model system Arabidopsis thaliana to elucidate plant defense signaling and pathway networks indicate that salicylic acid (SA) is the key hormone triggering the plant defense response against biotrophic and hemi-biotrophic pathogens, while jasmonic acid (JA) and derivatives are critical to the defense response against necrotrophic pathogens. Several reports demonstrate that SA limits nematode reproduction. Results Here we translate knowledge gained from studies using Arabidopsis to soybean. The ability of thirty-one Arabidopsis genes encoding important components of SA and JA synthesis and signaling in conferring resistance to soybean cyst nematode (SCN: Heterodera glycines) are investigated. We demonstrate that overexpression of three of thirty-one Arabidoposis genes in transgenic soybean roots of compost plants decreased the number of cysts formed by SCN to less than 50% of those found on control roots, namely AtNPR1(33%), AtTGA2 (38%), and AtPR-5 (38%). Three additional Arabidopsis genes decreased the number of SCN cysts by 40% or more: AtACBP3 (53% of the control value), AtACD2 (55%), and AtCM-3 (57%). Other genes having less or no effect included AtEDS5 (77%), AtNDR1 (82%), AtEDS1 (107%), and AtPR-1 (80%), as compared to control. Overexpression of AtDND1 greatly increased susceptibility as indicated by a large increase in the number of SCN cysts (175% of control). Conclusions Knowledge of the pathogen defense system gained from studies of the model system, Arabidopsis, can be directly translated to soybean through direct overexpression of Arabidopsis genes. When the genes, AtNPR1, AtGA2, and AtPR-5, encoding specific components involved in SA regulation, synthesis, and signaling, are overexpressed in soybean roots resistance to SCN is enhanced. This demonstrates functional compatibility of some Arabidopsis genes with soybean and identifies genes that may be used to engineer resistance to nematodes. [ABSTRACT FROM AUTHOR]

Farnesol is an isoprenoid intermediate in the mevalonate (MVA) pathway and is produced by the dephosphorylation of farnesyl diphosphate. Farnesol plays a central role in cell growth and differentiation, controls production of ubiquinone and ergosterol, and participates in the regulation of filamentation and biofilm formation. Despite these important functions, studies of farnesol in filamentous fungi are limited, and information on its effects on antifungal and/or biocontrol activity is scarce. In the present article, we identified the Trichoderma harzianum gene dpp1 , encoding a diacylglycerol pyrophosphatase that catalyzes production of farnesol from farnesol diphosphate. We analyzed the function of dpp1 to address the importance of farnesol in Trichoderma physiology and ecology. Overexpression of dpp1 in T. harzianum caused an expected increase in farnesol production as well as a marked change in squalene and ergosterol levels, but overexpression did not affect antifungal activity. In interaction with plants, a dpp1 -overexpressing transformant acted as a sensitizing agent in that it up-regulated expression of plant defense salicylate-related genes in the presence of a fungal plant pathogen. In addition, toxicity of farnesol on Trichoderma and plants was examined. Finally, a phylogenetic study of dpp1 was performed to understand its evolutionary history as a primary metabolite gene. This article represents a step forward in the acquisition of knowledge on the role of farnesol in fungal physiology and in fungus-environment interactions.

Abstract: Purpose: Overexpression of proto-oncogene HER-2 is one of the main molecular markers of breast cancer involved in prognosis and diagnosis and also in trastuzumab therapy. Thus, a request for the evaluation of HER-2 status in breast cancer has been increasing. The aim of our study was assessment of HER-2 overexpression in malignant and benign breast cancer specimens by Real Time RT-PCR technique and comparison of its results with IHC outcomes. Methods: Twenty benign and sixty malignant breast cancers in addition to fifteen normal breast tissue specimens were analyzed by Real Time RT-PCR method. Fresh tissue samples were disrupted by mortar and pestle. A syringe and a needle were used for complete homogenization of the tissues. The RNA was then isolated from the samples and converted to cDNA. A standard curve was initially plotted using BioEasy SYBR Green I and then all 95 specimens were studied by Real Time RT-PCR using 2−ΔΔCt method. Results: 23.3% of 60 malignant specimens showed HER-2 overexpression, while all of the benign samples represented the normal expression level of HER-2 gene. The concordance rate between the results of Real Time RT-PCR and IHC was 86.6%. Conclusion: Real Time RT-PCR method is an almost reliable technique and at least can be used as a complementary method for confirming IHC results. This is emanated from relatively high rate of concordance between outcomes of IHC test, as a routine method of detecting the HER-2 gene expression status, and Real Time RT-PCR technique. [Copyright &y& Elsevier]

The PmPR10-1.10 protein from western white pine is known to be associated with frost hardiness, and up-regulated by seasonal cold acclimation and biotic and abiotic stresses. To gain insight into the molecular basis of cold hardiness, we investigated the potential physiological role of PmPR10-1.10 by gene overexpression in transgenic Arabidopsis plants. A binary vector was constructed for PmPR10-1.10 synthesis in higher plants and transgenic Arabidopsis lines were generated by Agrobacterium-mediated transformation. Following Western protein blot analysis confirming target protein production, transgenic Arabidopsis lines were tested for cold tolerance by electrolyte leakage analysis post treatment of different freezing temperatures. Our results demonstrate that accumulation of PmPR10-1.10 protein resulted in significantly greater freezing tolerance in transgenic plants than in wild type plants. This indicates that the transfer and selection of cold acclimation proteins like PmPR10-1.10 may be a breeding strategy for the development of freezing tolerance in conifers. [ABSTRACT FROM AUTHOR]

By exploring microarray data in a salt-tolerant wheat mutant, RH8706-49, we identified a novel stress-related gene, namedTaSTG(Triticum aestivumsalt tolerance gene), which can be induced by salt stress in wheat. Transgenic rice andArabidopsisplants overexpressingTaSTGwere generated and proved to be more salt-tolerant than the wild-type control. Seeds of transgenicArabidopsisexhibited an elevated germination rate under salt stress. Compared with those of the wild type, the levels of chlorophyll, soluble sugars and proline in transgenicArabidopsiswere significantly higher, whereas the levels of Na+and malondialdehyde were significantly lower. Moreover, the expression profile of seven salt tolerance-related genes, alcohol dehydrogenase class-P (ADH1), cold-regulated protein 15a (COR15A), inositol polyphosphate 1-phosphatase FIERY1 (FRY1), Gamma-glutamyl phosphate reductase (P5CS1), desiccation-responsive rd29 (RD29B), U6 snRNA-associated Sm-like protein (SAD1), salt overly sensitive 3 (SOS3), in transgenicArabidopsissuggests their possible interaction withTaSTG. Compared with those of the wild type, the expression levels of six of these genes in transgenic plants were significantly increased, whereas the expressionof SOS3was decreased. We propose thatTaSTGmay act upstream of the up-regulated genes, possibly by regulating plant responses to osmotic stress through phospholipases and CDPK pathway, consequently enhancing plant salt tolerance via proline accumulation. [ABSTRACT FROM AUTHOR]

Plants require a complex balance of mineral nutrients to reproduce successfully. Because the availability of many of these nutrients in the soil is compromised by several factors, such as soil pH, cation presence, and microbiai activity, crop plants depend directly on nutrients applied as fertilizers to achieve high yields. However, the excessive use of fertilizers is a major environmental concern due to nutrient leaching that causes water eutrophication and promotes toxic algae blooms. This situation generates the urgent need for crop plants with increased nutrient use efficiency and better-designed fertilization schemes. The plant biology revolution triggered by the development of efficient gene transfer systems for plant cells together with the more recent development of next-generation DNA and RNA sequencing and other omics platforms have advanced considerably our understanding on the molecular basis of plant nutrition and how plants respond to nutritional stress. To date, genes encoding sensors, transcription factors, transporters, and metabolic enzymes have been identified as potential candidates to improve nutrient use efficiency. In addition, the study of other genetic resources, such as bacteria and fungi, allows the identification of alternative mechanisms of nutrient assimilation, which are potentially applicable in plants. Although significant progress in this respect has been achieved by conventional breeding, in this review we focus on the biotechnological approaches reported to date aimed at boosting the use of the three most limiting nutrients in the majority of arable lands: nitrogen, phosphorus, and iron. [ABSTRACT FROM AUTHOR]

Histidine biosynthesis in Corynebacterium glutamicum is regulated not only by feedback inhibition by the first enzyme in the pathway, but also by repression control of the synthesis of the histidine enzymes. C. glutamicum histidine genes are located and transcribed in two unlinked loci, hisEG and hisDCB-orf1-orf2-hisHA-impA-hisFI. We constructed plasmid pK18 hisDP tac to replace the native hisD promoter with the tac promoter, and overexpressed phosphoribosyl-ATP-pyrophosphohydrolase, encoded by hisE, and ATP-phosphoribosyltransferase, encoded by hisG. The l-histidine titer at 0.85 g l was 80 % greater in the transformed bacterium and production of byproducts, l-alanine and l-tryptophan, was significantly decreased. However, accumulation of glutamic acid increased by 58 % (2.8 g l). This study represents the first attempt to substitute the histidine biosynthesis pathway promoter in the chromosome with a stronger promoter to increase histidine production. [ABSTRACT FROM AUTHOR]

Abstract: Trypanosoma cruzi calreticulin (TcCRT) can hijack complement C1, mannan-binding lectin and ficolins from serum thus inhibiting the classical and lectin complement pathway activation respectively. To understand the in vivo biological functions of TcCRT in T. cruzi we generated a clonal cell line lacking one TcCRT allele (TcCRT+/−) and another clone overexpressing it (TcCRT+). Both clones were derived from the TCC T. cruzi strain. As expected, TcCRT+/− epimastigotes showed impairment on TcCRT synthesis, whereas TcCRT+ ones showed increased protein levels. In correlation to this, monoallelic mutant parasites were significantly susceptible to killing by the complement machinery. On the contrary, TcCRT+ parasites showed higher levels of resistance to killing mediate by the classical and lectin but not the alternative pathway. The involvement of surface TcCRT in depleting C1 was demonstrated through restoration of serum killing activity by addition of exogenous C1. In axenic cultures, a reduced propagation rate of TcCRT+/− parasites was observed. Moreover, TcCRT+/− parasites presented a reduced rate of differentiation in in vitro assays. As shown by down- or upregulation of TcCRT expression this gene seems to play a major role in providing T. cruzi with the ability to resist complement system. [Copyright &y& Elsevier]

Abstract: Purpose: To examine the effects of oxymatrine (OMT) on ventricular remodeling in spontaneous hypertension rat (SHR) and the underlying mechanism. Methods: SHRs were divided into four groups: SHR control, SHR+40mg/kg captopril, SHR+30mg/kg OMT and SHR+60mg/kg OMT. Normotensive age-matched WKY rats were assigned to two groups: WKY control, WKY+30mg/kg OMT. The rats were orally administered with the corresponding drugs or drinking water for 21 weeks. Mean arterial blood pressure (MAP) and heart rate (HR) were measured. The left ventricular weight index (LVWI) and heart weight index (HWI) were determined. Myocardium tissue was stained with picric acid/Sirius red for measurement of collagen content measurements. The concentrations of serum norepinephrine and angiotensin II (Ang II) in myocardium were determined. Real-time RT-PCR was used to detect the mRNA expressions of transforming growth factor-β1 (TGF-β1), collagen types I, III and angiotensin converting enzyme (ACE). Western blots were performed to determine bioactivities of extracellular signal regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK/SAPK), p38 mitogen-activated protein kinases (p38 MAPK) and phospho-specific protein kinase C (PKC). Results: In the SHR, hypertension, myocardium hypertrophy, more cardiac fibrosis, higher concentrations of serum norepinephrine and myocardium Ang II were observed. OMT treatment lowered the blood pressure, reduced the concentrations of serum norepinephrine and myocardium Ang II, favorably decreased the measured gravimetric parameters, decreased the interstitial and perivascular collagen deposition, attenuated the collagen of type I and III accumulation, downregulated the mRNA expression of ACE and TGF-β1, and suppressed the phosphorylation of ERK 1/2, JNK and p38 MAPK in SHRs. Conclusion: OMT prevents ventricular remodeling in SHR. The mechanisms may be related to inhibiting the gene overexpression of ACE and TGF-β1, suppressing the activation of signaling pathways of ERK 1/2, JNK and p38 MAPK. [Copyright &y& Elsevier]

Gene overexpression has been identified as a primary determining factor for the distinct Down syndrome (DS) phenotypes. Previous genetic research has identified a spectrum of gene expressions responsible for many of the observed traits in DS patients including cardiovascular, brain, and GI anomalies. However, the molecular/genetic basis underlying pulmonary anomalies are yet to be identified, even though respiratory complications represent the leading cause of morbidity and mortality in DS patients. In this article, we will discuss the Etiopathogenesis and spectrum of pulmonary anomalies in DS patients. [ABSTRACT FROM AUTHOR]

The aim of this study is to elucidate the relation between expression of coxsackie and adenovirus receptor (CAR) and formation of lung cancer. We investigated the expression of CAR by immunohistochemistry, Western blot and real-time RT-PCR in 120 lung cancers. We found that CAR expression in tumor tissues was significantly higher than that in normal lung tissues. CAR expression had a correlation with the histological grade of lung squamous cell carcinoma; however, there was no relationship between the CAR expression and the other clinical pathological features. In vitro, silencing or overexpression of CAR could significantly inhibit or promote colony formation, cell adhesion, and invasion in A549 cells. Our findings demonstrated that CAR may play an essential role in the formation of lung cancer. [ABSTRACT FROM AUTHOR]

Edeines are a group of non-ribosomal antibacterial peptides produced by Brevibacillus brevis . Due to the significant antibacterial properties of edeines, increasing edeine yield is of great interest in biomedical research. Herein, we identified that EdeB, a member of the ParB protein family, significantly improved edeine production in B. brevis . First, overexpression of edeB in B. brevis X23 increased edeine production by 92.27%. Second, in vitro bacteriostasis experiment showed that edeB -deletion mutant exhibited less antibacterial activity. Third, RT-qPCR assay demonstrated that the expression of edeA , edeQ , and edeK , which are key components of the edeine biosynthesis pathway, in edeB -deletion mutant X23(ΔedeB) was significantly lower than that in wild-type B. brevis strain X23. Finally, electrophoretic mobility shift assay (EMSA) showed that EdeB directly bound to the promoter region of the edeine biosynthetic gene cluster ( ede BGC), suggesting that EdeB improves edeine production through interaction with ede BGC in B. brevis ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Du, Zhang, Long, Zhang, Chen and Liu.)

Microalgal lipids are essential for biofuel and dietary supplement production. Lipid engineering for higher production has been studied for years. However, due to the complexity of lipid metabolism, single-gene engineering gradually encounters bottlenecks. Multiple gene regulation is more beneficial to boosting lipid accumulation and further clarifying the complex regulatory mechanism of lipid biosynthesis in the homeostasis of lipids, carbohydrates, and protein metabolism. Here, three lipid-related genes, DOF, LACS2, and CIS, were co-regulated in Chlamydomonas reinhartii by two circles of transformation to overexpress DOF and knock down LACS2 and CIS simultaneously. With the multiple regulations of these genes, the intracellular lipids and FA content increased by 142% and 52%, respectively, compared with CC849, whereas the starch and protein contents decreased by 45% and 24%. Transcriptomic analysis showed that genes in TAG and FA biosynthesis were up-regulated, and genes in starch and protein metabolism were down-regulated. This revealed that more carbon precursor fluxes from starch and protein metabolism were redirected towards lipid synthesis pathways. These results showed that regulating genes in various metabolisms contributed to carbon flux redirection and significantly improved intracellular lipids, demonstrating the potential of multiple gene regulation strategies and providing possible candidates for lipid overproduction in microalgae.

Gossypium hirsutum L. represents the best cotton species for fiber production, thus computing the largest cultivated area worldwide. Meloidogyne incognita is a root-knot nematode (RKN) and one of the most important species of Meloidogyne genus, which has a wide host range, including cotton plants. Phytonematode infestations can only be partially controlled by conventional agricultural methods, therefore, more effective strategies to improve cotton resistance to M. incognita disease are highly desirable. The present study employed functional genomics to validate the involvement of two previously identified candidate genes, encoding dirigent protein 4- GhDIR4 and peroxiredoxin-2- GhPRXIIB , in cotton defense against M. incognita . Transgenic A. thaliana plant lines overexpressing GhDIR4 and GhPRXIIB genes were generated and displayed significantly improved resistance against M. incognita infection in terms of female nematode abundance in the roots when compared to wild-type control plants. For our best target-gene GhDIR4 , an in-silico functional analysis, including multiple sequence alignment, phylogenetic relationship, and search for specific protein motifs unveiled potential orthologs in other relevant crop plants, including monocots and dicots. Our findings provide valuable information for further understanding the roles of GhDIR and GhPRXIIB genes in cotton defense response against RKN nematode., Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03282-4., Competing Interests: Conflict of interestsThe authors declare that the research was conducted in the absence of any commercial or financial competing interest., (© King Abdulaziz City for Science and Technology 2022, Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)